The type III secretion system of Gram-negative bacterial pathogens creates one of the most direct interfaces between pathogens and their hosts. These 'needle-like'molecular machines inject bacterial effector proteins directly into host cells for the purpose of destroying an innate immune response and facilitating bacterial replication, dissemination, and disease progression. Effector proteins are unique virulence factors in that they often capture or mimic the properties of host signal transduction molecules. One such target is the evolutionarily conserved Ras-superfamily of GTPases. The present study focuses on a recently identified bacterial type III effector family. The related type III effectors SifA, IpgB, and Map are required for Salmonella, Shigella, and enterohaemorrhagic E. coli pathogenesis, respectively, through their common ability to activate Rho-family GTPase signaling cascades. The studies described here seek to elucidate the host signaling mechanisms of this large bacterial virulence factor family by examining 1) the enzymatic activation of Rho GTPases, 2) type III effector recognition of GTPases at the molecular level, and 3) the effects of effector protein localization within the host cell. By revealing mechanistic details of type III effector family members, these studies will provide new insights into the pathogenic mechanisms o several infectious agents and into the biology of their human host. PUBLIC HEALTH RELEVANCE: Human Rho-family GTPases are major targets of bacterial toxins and effector proteins. Pathogens hijack this critical signaling pathway to facilitate bacterial replication, dissemination, and disease progression. This proposal examines the ability of a large family of bacterial type III effector proteins to hijack human Rho GTPases. A deeper understanding of the enzymatic and biochemical interface between these bacterial effectors and human GTPases will lead to a more complete knowledge of numerous pathogenic mechanisms and may reveal new aspects of signal transduction in the human host cell.